The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
John S. Olson - One of the best experts on this subject based on the ideXlab platform.
-
Myoglobin as a model system for designing heme protein based blood substitutes
Biophysical Chemistry, 2002Co-Authors: Yi Dou, David H Maillett, Raymund F Eich, John S. OlsonAbstract:Abstract The ligand binding properties and resistances to denaturation of >300 different site-directed mutants of sperm whale, pig, and human Myoglobin have been examined over the past 15 years. This library of recombinant proteins has been used to derive chemical mechanisms for ligand binding and to examine the factors governing holo- and apoglobin stability. We have also examined the effects of mutagenesis on the dioxygenation of NO by MbO 2 to form NO 3 − and metMb. This reaction rapidly detoxifies NO and is a key physiological function of both Myoglobins and hemoglobins. The mechanisms derived for O 2 binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O 2 delivery pharmaceuticals in transfusion therapy (i.e. blood substitutes). An interactive database is being developed ( http://olsonnt1.bioc.rice.edu/web/Myoglobinhome.asp ) to allow rapid access to the ligand binding parameters, stability properties, and crystal structures of the entire set of recombinant Myoglobins. The long-range goal is to use this library for developing general protein engineering principles and for designing individual heme proteins for specific pharmacological and industrial uses.
-
high resolution crystal structures of distal histidine mutants of sperm whale Myoglobin
Journal of Molecular Biology, 1994Co-Authors: Michael L Quillin, John S. Olson, Robert M Arduini, George N PhillipsAbstract:The highly conserved distal histidine residue (His64) of sperm whale Myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. MetMyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amine acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxyMyoglobin mutants. The water molecule that is hydrogen-bonded to the Ne atom of histidine 64 in wild-type deoxyMyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 Myoglobins in the P 6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 Myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in Myoglobin.
-
genetic engineering of Myoglobin as a simple prototype for hemoglobin based blood substitutes
Artificial Cells Blood Substitutes and Biotechnology, 1994Co-Authors: John S. OlsonAbstract:Site-directed mutagenesis has been used to examine the structural and functional roles of distal pocket residues in regulating O2 affinity, CO binding, rates of association and dissociation, autooxidation, and hemin loss in mammalian Myoglobins and human hemoglobin. In Myoglobin, His-E7 inhibits CO binding by requiring displacement of distal pocket water. In the case of O2 binding, this displacement is compensated by a strong hydrogen bond between the bound ligand and the imidazole side chain. The isopropyl side chain Val-E11 also sterically restricts CO binding. The rates of ligand binding are regulated by distal pocket water displacement, steric restrictions near the iron atom, and an outer more global protein barrier. Autooxidation occurs by two mechanisms, direct dissociation of HO2 and bimolecular reaction of external O2 with unliganded heme. Both processes are inhibited markedly by hydrogen bonding interactions with His-E7. Double mutants have been constructed to decrease oxygen affinity, but still ...
-
the mechanism of autooxidation of Myoglobin
Journal of Biological Chemistry, 1993Co-Authors: R E Brantley, E W Singleton, Anthony J. Wilkinson, Stephen J Smerdon, John S. OlsonAbstract:Abstract Time courses for the autooxidation of native and mutant sperm whale and pig Myoglobins were measured at 37 degrees C in the presence of catalase and superoxide dismutase. In sperm whale Myoglobin, His64(E7) was replaced with Gln, Gly, Ala, Val, Thr, Leu, and Phe; Val68(E11) was replaced with Ala, Ile, Leu, and Phe; Leu29(B10) was replaced with Ala, Val, and Phe. In pig Myoglobin, His64(E7) was replaced with Val; Val68(E11) was replaced with Thr and Ser; Thr67(E10) was replaced with Ala, Val, Glu, and Arg; Lys45(CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo-first order rate constants varied over 4 orders of magnitude, from 58 h-1 (H64A) to 0.055 h-1 (native) to 0.005 h-1 (L29F) at 37 degrees C, pH 7, in air. The dependences of the observed autooxidation rate constant on oxygen concentration and pH were measured for native and selected mutant Myoglobins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high [O2], direct dissociation of the neutral superoxide radical (HO2) from oxyMyoglobin dominates, and this process is accelerated by decreasing pH. At low [O2], autooxidation occurs by a bimolecular reaction between molecular oxygen and deoxyMyoglobin containing a weakly coordinated water molecule. The neutral side chain of the distal histidine (His64) inhibits autooxidation by hydrogen bonding to bound oxygen, preventing both HO2 dissociation and the oxidative bimolecular reaction with deoxyMyoglobin. Replacement of His64 by amino acids incapable of hydrogen bonding to the bound ligand markedly increases the rate of autooxidation and causes the superoxide mechanism to predominate. Increasing the polarity of the distal pocket by substitution of Val68 with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II).O2 complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.
-
contributions of residue 45 cd3 and heme 6 propionate to the bimolecular and geminate recombination reactions of Myoglobin
Biochemistry, 1991Co-Authors: Theodore E Carver, John S. Olson, Quentin H. Gibson, Anthony J. Wilkinson, Stephen J Smerdon, Szymon Krzywda, Richard S Blackmore, Dezz J Ropp, Stephen G SligarAbstract:: Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale Myoglobins and to sperm whale Myoglobin reconstituted with protoheme IX dimethyl ester. In pig Myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale Myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig Myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of Myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of Myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig Myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)
Kevin P Moore - One of the best experts on this subject based on the ideXlab platform.
-
a causative role for redox cycling of Myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis induced renal failure
Journal of Biological Chemistry, 1998Co-Authors: Kevin P Moore, Steve Holt, Rakesh P Patel, Dimitri A Svistunenko, William Zackert, David Goodier, Brandon J Reeder, Martine Clozel, Radhi Anand, Chris E CooperAbstract:Abstract Muscle injury (rhabdomyolysis) and subsequent deposition of Myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that Myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), Myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of Myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by ∼80%. EPR and UV spectroscopy demonstrated that Myoglobin was deposited in the kidneys as the redox competent ferric Myoglobin and that it’s concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl Myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that Myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of Myoglobin-induced lipid peroxidation.
-
a causative role for redox cycling of Myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis induced renal failure
Journal of Biological Chemistry, 1998Co-Authors: Kevin P Moore, Steve Holt, Rakesh P Patel, Dimitri A Svistunenko, William Zackert, David Goodier, Brandon J Reeder, Martine Clozel, Radhi Anand, Chris CooperAbstract:Muscle injury (rhabdomyolysis) and subsequent deposition of Myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that Myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), Myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of Myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that Myoglobin was deposited in the kidneys as the redox competent ferric Myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl Myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that Myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of Myoglobin-induced lipid peroxidation.
Stephen G Sligar - One of the best experts on this subject based on the ideXlab platform.
-
contributions of residue 45 cd3 and heme 6 propionate to the bimolecular and geminate recombination reactions of Myoglobin
Biochemistry, 1991Co-Authors: Theodore E Carver, John S. Olson, Quentin H. Gibson, Anthony J. Wilkinson, Stephen J Smerdon, Szymon Krzywda, Richard S Blackmore, Dezz J Ropp, Stephen G SligarAbstract:: Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale Myoglobins and to sperm whale Myoglobin reconstituted with protoheme IX dimethyl ester. In pig Myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale Myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig Myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of Myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of Myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig Myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)
-
analysis of the kinetic barriers for ligand binding to sperm whale Myoglobin using site directed mutagenesis and laser photolysis techniques
Journal of Biological Chemistry, 1990Co-Authors: Theodore E Carver, John S. Olson, Quentin H. Gibson, Richard S Blackmore, Ronald J Rohlfs, Barry A Springer, Stephen G SligarAbstract:Abstract Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale Myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the Myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native Myoglobin. For Ile68 Myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native Myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant Myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into Myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into Myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)
-
the effects of amino acid substitution at position e7 residue 64 on the kinetics of ligand binding to sperm whale Myoglobin
Journal of Biological Chemistry, 1990Co-Authors: Ronald J Rohlfs, John S. Olson, Theodore E Carver, Barry A Springer, Antony J Mathews, Karen D Egeberg, Stephen G SligarAbstract:Abstract Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale Myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered Myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant Myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately Gln64 (10(5)-10(3) M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64----Arg and His64----Asp mutants were abnormally high (1-5 x 10(6) M-1 s-1), suggesting that Arg64 and Asp64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64----Gln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for Myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native Myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxyMyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64----Gln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant Myoglobin (Gln-E7), Aplysia limacina Myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).
Chris E Cooper - One of the best experts on this subject based on the ideXlab platform.
-
a causative role for redox cycling of Myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis induced renal failure
Journal of Biological Chemistry, 1998Co-Authors: Kevin P Moore, Steve Holt, Rakesh P Patel, Dimitri A Svistunenko, William Zackert, David Goodier, Brandon J Reeder, Martine Clozel, Radhi Anand, Chris E CooperAbstract:Abstract Muscle injury (rhabdomyolysis) and subsequent deposition of Myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that Myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), Myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of Myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by ∼80%. EPR and UV spectroscopy demonstrated that Myoglobin was deposited in the kidneys as the redox competent ferric Myoglobin and that it’s concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl Myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that Myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of Myoglobin-induced lipid peroxidation.
Chris Cooper - One of the best experts on this subject based on the ideXlab platform.
-
a causative role for redox cycling of Myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis induced renal failure
Journal of Biological Chemistry, 1998Co-Authors: Kevin P Moore, Steve Holt, Rakesh P Patel, Dimitri A Svistunenko, William Zackert, David Goodier, Brandon J Reeder, Martine Clozel, Radhi Anand, Chris CooperAbstract:Muscle injury (rhabdomyolysis) and subsequent deposition of Myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that Myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), Myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of Myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that Myoglobin was deposited in the kidneys as the redox competent ferric Myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl Myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that Myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of Myoglobin-induced lipid peroxidation.
